Background: Extracellular vesicles (EVs) in blood prod­ ucts are potential effectors of inflammation and coag­ ulation after transfusion. The aim of this study was to assess the impact of different blood manufacturing methods and duration of hypothermic storage on the EV subpopulations in relation to other in vitro quality pa­ rameters of red blood cell concentrate (RCC) products.Methods: RCCs were produced using whole blood filtra­ tion (WBF) or red cell filtration (RCF) (n = 12/method), re­ frigerated for 43 days, and evaluated for EV size profile and concentration, red cell deformability, ATP and 2,3­ DPG, hemolysis, and hematological indices. Results: The total number of EVs increased significantly with storage in both methods, and WBF­RCCs contained the higher numbers of EVs compared to RCF­RCCs. The concentra­ tion of small EVs was greater in WBF­RCCs versus RCF­ RCCs, with difference between the two methods ob­ served on day 43 of storage (p = 0.001). Throughout stor­ age, significant decreases were identified in ATP, 2,3­ DPG, and EImax, while an increase in hemolysis was observed in both RCC products. Conclusion: The dy­ namic shift in the size and concentration of the EV sub­ populations is dependent on the blood manufacturing method and length of storage. Better understanding of the potential clinical implications of these heterogene­ ous populations of EVs are needed.